just follow Koch's postulates

Hi Fatemah: First thing you should do surf sterilize with bleach 10% for 1-2 min and after that pace it on PDA media petridish, incubated in 25 C  for 5 day and check what you get from it, and you can pure what you get in new PDA dish with hyphal tip techniqe,  this for fungal isolation

For bacteria isolation you can do the same with fungal isolation but grind the plant tissue after sterilize on glass slide, with a loop take swipe and zig-zag on NA media and incubated in 37 C for 48 h,    

I have understood that the  diseases in animals is due to inadequate nutritinal intake; i.e., an ancient  Greek  temple  of  Apollo  at  Delphi  (BC  8  C)  has  borne  the

inscription  “Meden  Agan  (μηδὲν  ἄγαν;  nothing  in  excess)”,  and  such  ancient  philosophers  as Aristotle (Greece; BC 4 C), Confucius (China; BC 6 C), and Gotama Siddhattha (India; BC 6 C) have already indicated about the importance of “Golden or Happy Mean” (file Hara-hachibun-me).  This seems also applicable onto plants; i.e., opportunistic infections also seem to occur in plants. 

Furthermore, I have recently found that animal tissues and cells contain invading or coexisting fungal, bacterial, plant viral, plant proteins as determined by PDMD method (file HepG2 fucoidan).  I have demonstrated that HIV and HCV have  invaded or co-infected into the HepG2 hepatoma cells.   Therefore, I would like to recommend the application of PDMD method also onto the plant pathology.

Hi: First thing you should isolate the pathogen in media culture, for Fungal use PDA media and for bacteria use NA. from the symptom on the plant you can decide is it fungal or bacterial disease. when the pathogen grow in media you can purify in pure culture by taken hyphal tip of fungal colony an culture it in new PDA petridish and for bacteria take wisp by loop from single colony and zig-zag in new NA petridish

Thanks Dr. Oadi  for your answer. How can I decide by symptoms either it is bacteria or fungus?

Could you please add some references?

Thanks

Hello ¡

To determine the causal organism of a disease is not an easy task and need some specialization. Although you can follow all the procedures nicely described by Dr. Oadi and do by yourself, in my opinion at least till you acquire certain training from a Plant Pathology lab, you should consult a Plant Pathologist from your University or other Institutions close to your working place.

Regards,

Juan

Hi Fatemah

as Dr Juan suggested you should try to take help from a plant pathologist, but if you want to do it by yourself, its not much difficult.

First just took the infected tissue (Plant) surface sterilize it either by mercuric chloride or NaOCl, and grow the pathogen in a medium.

But now question is which medium and how you would get to know about the pathogen, is pathogen a fungi or bacteria? 

So to resolve this question, first just superficially observe the infected tissue, is there any fungal mycelium and other fungal structure is visible or bacterial colonies/spot. it will help you. but in any case, as you are not sure that pathogen is bacteria or fungi, just allow the pathogen to grow in PDA and Nutrient Agar medium. you will get many bacterial and fungal colonies. purify those colonies by serial subculturing and with the pure cultures, perform the Koch postulates.

Koch postulates will prove which pathogen is involved in disease development.

Once you get information about the Pathogen, you can go for its identification further. 

any further help, you are most welcome.

Wish you luck.

 Thanks all for your answers.

Can I get a reference for that protocols?

You need to proof compliance with Koch's postulates, which are as follows:

1.) The microorganism must be found in symptomatic plants, but not in healthy plants.

2.) The microorganism must be isolated from a diseased plant and grown in pure culture.  There are a few exceptions to the latter half of this rule for non-culturable pathogens.

3.) The cultured microorganism should cause disease with the same symptoms when inoculated onto a healthy organism.

4.) The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Others have given you guidance as to how this is best done